The small intestinal lining cell (enterocyte) represents a unique model for studying the mechanisms involved in regulating gene expression: (a) it is known to synthesize a broad spectrum of well characterized proteins; (b) its differentiation is rapidytopographically well organized and occurs continuously and; (c) it must respond to a wide variety of luminal (i.e., dietary) as well as antiluminal (e.g. hormonal) stimuli. Despite this potential for genetic analysis, the mRNA population of the enterocyte has not been well described. This proposal is designed to study the regulation of apolipoprotein gene expression in human and rat enterocytes. This system was selected because (i) the intestine is an important site for apolipoprotein synthesis, (ii) some of the apolipoprotein mRNAs are abundant, comprising 1% of the mRNA population of small intestinal epithelium, (iii) apolipoproteins have been characterized with respect to their structure and function, (iv) as components of lipoproteins they play a central role atherogenesis, and (v) metabolic/genetic lipoprotein abnormalities occur in man. Specific methods used include translation of intestinal mRNA in cell-free systems and cloning cDNAs encoding all, or a portion, of apolipoproteins AI, AII, AIV, and B. In order to characterize the primary translation products of these mRNAs, the NH2-termini of the in vitro products will be sequenced using automated sequential Edman degradation and the results compared with the NH2-terminal structure of apolipoproteins isolated from HDL and LDL. Since we have found that some of these apolipoproteins are synthesized as preproteins, cotranslational and posttranslational cleavage with signal peptidase will be performed and the results related to in vivo lipoprotein assembly. RNA excess hybridization analyses using cloned cDNAs will be used to quantitate specific mRNA accumulation within the intestinal epithelium after a variety of metabolic perturbations as well as to determine the degree of coordinate expression of these genes. cDNAs will also be used to correlate specific apolipoprotein gene expression with enterocyte maturation and to define apolipoprotein mRNA kinetics in states of malabsorption and altered lipoprotein metabolism. It should be possible to construct a molecular paradigm for the intestinal contribution to dysapolipoproteinemias.